Antiviral Activity Inside Host Cells
Blocking the viral entry, via liquid/surface disinfection or once in the body, is a powerful strategy to hamper early stage viral contagions. On the other hand, when infections have already spread and reached middle and late stages, alternative pharmacological strategies are required. The study of the viral pathogenesis and machinery inside host cells has allowed the preparation of different drugs. Due to the present pandemic, such antiviral drugs are now of top interest in the scientific and medical communities. So far, few antiviral drugs are clinically available, and their mechanisms of action consist on the inhibition of reverse transcriptase (HIV, hepatitis), DNA inhibition of polymerase (herpes, HIV), inhibition of protease (HIV), blockage of ion channels (influenza), and inhibition of neuramidase (HIV, influenza, hepatitis). However, these drugs suffer from moderate to severe side effects. Additionally, rapid mutations in the viral machinery make them resistant to the treatments, making the control and the stop of the infection challenging. The use of HNMs in drug delivery has shown several advantages. First, HNMs can increase drug solubility and its circulation time. Additionally, they can be functionalized with targeting molecules able to direct the drug to the desired organs and so avoiding side effects and reducing the dosage. More recently, new antiviral mechanisms were discovered. In particular, it was found that different types of HNMs are able to change the ROS homeostasis in infected host cells, stopping the viral replication and preserving the cell survival. In this section both drug delivery materials and HNMs with ROS modulation properties will be critically presented (Figure 10).
Figure 10  Illustration of NMs interacting with host cell to prevent viral spreading. Left: NMs are used for delivery of antiviral agents. Right: NMs can change the ROS homeostasis slowing down the infection and helping cell survival.

Nanomaterials for Drug Delivery
Different HNMs have been widely tested for drug delivery applications. The advantages of this strategy are several including enhance drug solubility and the possibility of targeting and multivalent effects. As a potential broad spectral antiviral agent, AgNPs can prevent the virus from adsorbing to the host cell in the early stage of infection and thus show strong antiviral activity. After the cells are infected by the virus, there are ways to inhibit cell apoptosis (Figure 2, right). For example, Lv et al. studied the anti-TGEV activity of AgNPs in swine testicle cells and explored the possible mechanism of AgNP inhibition of TGEV infection-induced apoptosis.18 The results showed that these AgNPs are able to decrease cell apoptosis through the activation of p38/mitochondria-caspase-3 signaling.18
Although the use of AgNPs has shown good antiviral action to further improve the therapeutic effects and reduce both side effects and drug resistance, the way of binding drugs or genes and other therapeutic agents to AgNPs has received particular attention. It has been observed that AgNPs inhibit the activities of neuraminidase and hemagglutinin, preventing the H1N1 influenza virus from attaching to host cells. At the same time, the potential molecular mechanisms revealed that caspase-3-mediated apoptosis was inhibited by ROS generation. AgNPs modified with polyethylenimine (PEI) can bind siRNA. Ag@PEI@siRNA exhibited superior abilities for enhanced cellular uptake and blocking EV71 virus infection and significantly decreased the apoptotic cell population, which prevented the spread of EV71 virus.37 In addition to drugs and siRNA, neutralizing antibodies in combination with AgNPs were developed. The results demonstrated that there is an additive effect between the antibody and AgNPs when combined against cell-associated HIV-1 infection in vitro.96
The membranotropic properties of fullerenes were widely exploited. For example, pristine C60, after accumulation at the cell membrane, can translocate into the cytoplasm by crossing the membrane through multiple energy-dependent pathways despite its hydrophobic character.97 Fullerenes can be made water dispersible using surfactants, sonication or first dissolving them in appropriate organic solvents (DMSO). The use of fullerenes as inhibitors of viruses started in 1993 with a study focused on HIV infection.98 This work revealed the interaction between C60 derivatives and HIV protease through molecular modeling and experimental verification. HIV protease (HIV-PR) is involved in the mechanism of replication in the maturation of HIV virion, cleaving newly synthesized proteins. The active site of HIV protease has a cavity of 10 Å closed to the diameter of C60 cage.99 Molecular docking and experiments on HIV-PR catalytic activity revealed a blockage of the active site of the enzyme by van der Waals interactions leading to an antiviral effect. The following studies were based on a structure–activity relationship between functionalized fullerenes and HIV-PR with the aim to increase the antiviral activity. The introduction of pyrrolidinium salts onto C60 was tested against the activity of HIV-1 strain.100 In a second study, C60 bearing two ammonium groups was applied against the activity of HIV-1 and HIV-2 strains.101 The results showed the importance of having two moieties in a precise position on the fullerene cage and the influence of the charge of the different salts sensibly increasing its antiviral activity. Further investigations using different functional groups (e.g., amino acid derivatives) were explored. C60 functionalized with aminobutyric acid and aminocaproic acid was able to inhibit HIV viral replication at subnanomolar concentrations.63 In another work, anionic and cationic pyrrolidinium salts and amino acid functionalized fullerene derivatives were used for the inhibition of HIV reverse transcriptase (HIV-RT). Fullerene compounds were compared to nevirapine, an available drug against HIV-RT, revealing a better inhibition compared to the pure drug.102 The antiviral property of carboxylated fullerenes was confirmed by another study.103 Results obtained in vitro on CEM cell line showed low toxicity and a submicromolar EC50 against HIV-1 and HIV-2 strain viral replication.
Several mechanisms of HIV protease inhibitors have been hypothesized and simulated. Some studies investigated the action of fullerene derivatives on viral replication cycle and the virus maturation (Figure 11).98,104 For the latter, it was suggested an impairment due to a strong interaction between fullerene and the immature capsid.105
Figure 11  Illustration of fullerene affecting the viral replication mechanism. Interaction between fullerene and viral protein blocking either the transcription or the translation in the viral replication. Other RNA viruses share ways of viral replication similar to HIV.106 C60 functionalized with an amino acid derivative was investigated against Hepatitis C RNA polymerase (HCV-RP).102 This essential enzyme for viral replication was inhibited in a submicromolar range, similarly to benzo-1,2,4-thiadiazine, a potent specific inhibitor of HCV-RP. Another publication highlighted the effect of a C70 poly(carboxylic acid) derivative on different strains of influenza virus (e.g., A, H1N1, H3N2, and B).107 The inhibition was comparable to Tamiflu, rimantadine, ribavirin, and amantadine, but the mechanism of action remains still unclear. These data emphasize that fullerenes C60 or C70 can be potent universal antiviral drugs for RNA enveloped viruses. However, clear elucidations of the mechanisms of action and in vivo studies are still a missing point.
Due to their high surface/weight ratio and capacity to pass through cell membranes, carbon nanotubes (CNTs) have been extensively explored for drug and gene delivery applications.108 CNTs have been also successfully used for the delivery of antiviral agents. Compared to pristine materials, oxidized CNTs (ox-CNTs) showed an inhibitory activity for HIV viruses per se.109 This effect has been associated with the oxygenated groups that increase the hydrophilicity and the colloidal stability of the material. The antiviral efficiency has been correlated to the ox-CNT interaction with host cells.109 However, it is not clear how and what kind of mechanism blocks the viral machinery in host cells. Different anchoring strategies have been explored to link antiviral drugs onto CNT surface. For instance, ox-CNTs were covalently linked to 2-amino-3-nitro-1-(3,5-dimethylbenzyl)-aniline (CHI360) and N-(2-aminophenyl-3-nitro-)-3,5-dimethylbenzenesulfonamide (CHI415), two active non-nucleoside reverse transcriptase inhibitors for HIV treatment.109 Following the covalent conjugation, only a moderate antiviral effect was observed compared to pure drugs, indicating that most probably the NM cell trafficking played a key role on the virucidal activity.109 In another study, ox-CNTs functionalized with cyclodextrin were used for the delivery of acyclovir (a prodrug inhibitor of the viral DNA polymerases) for the treatment of HSV-1. Preliminary results showed that when acyclovir was delivered via the nanotubes, the viral antireplicative effect was higher than the free drug.110 More recently, a similar approach was applied to herpes virus using cyclodextrin and PEI-functionalized CNTs for co-delivery of cidofovir and plasmid DNA. However, the antiviral effect of the materials was not explained, and the transfection effect was not satisfactory.111 Functionalized CNTs have been also used for delivery of ribavirin in vivo using grass carp as an animal model for the study of grass carp reovirus. ox-CNTs were first functionalized via amidation with BSA, and then ribavirin was covalently bound to the protein via esterification (Figure 12).112
Figure 12  Schematic procedure of the functionalization of single-walled CNTs (SWCNTs) and ribavirin. Reproduced with permission from ref (112). Copyright 2015, Elsevier B.V. In vivo tests demonstrated that, when ribovirin was shuttled by ox-CNTs, the antiviral efficiency was significantly increased without any evident toxicity and no significant changes in ROS-generating enzymatic activities. The use of CNTs in drug delivery is however still controversial.113
Graphene-based materials have been limitedly studied as antiviral drug delivery carriers. Only a few examples can be found in the literature. Graphene quantum dots (GQDs) were used for drug delivery of CHI360 and CHI415 and tested in vitro against HIV similarly to CNTs.109 Both the prepared GQD-CHI360 and GQD-CHI415 showed a high antiviral activity once into host cells, with low toxicity. GO has been also used for the delivery of DNAzyme into hepatic cells allowing to block the hepatitis C infection. This specific DNA single strand is able to recognize the viral mRNA and to silence its expression.114
Overall, carbon NMs proved to be interesting carriers for antiviral drugs. However, several questions need to be answered before their safe application as antiviral materials. Different reports have demonstrated that pristine CNTs display relevant toxicity for healthy cells, but by oxidation of the tubes, the side effects can be sensibly reduced.109 In addition, more investigations should be performed on CNT toxicity. These nanocarriers indeed are not “innocent delivery agents”, but they play a key role in drug internalization pathway and in host cell machinery that might be averse to the expected therapeutic effects.109 So far, CNT application in biological systems has been studied for 20 years. However, due to their possible toxicity, their real application in clinics seems to be steeper and difficult to achieve.112

Materials Tuning Reactive Oxygen Species
ROS homeostasis in infected cells has been studied for both RNA and DNA viruses. For instance, it was shown that infection of mice with influenza A decreased the concentration of lung glutathione and the antioxidant vitamin C, providing evidence that the viral infection was associated with oxidative stress in vitro as well as in vivo.115 Similarly, in HIV infection, induced oxidative stress in host T cells and high concentrations of antioxidants are able to slow down the cell-to-cell viral spreading.115 The increase of ROS concentration is a common process in most of the viral infections. However, the mechanism of radical generation is different from case to case. Several proofs suggest that modulating ROS homeostasis in infected cells can slow down or block the infection.116
HNMs have been shown to be powerful allies against viral infections. In particular, metal or metal oxide NMs, once internalized, can regulate the radical production into infected host cells. In this scenario, the NMs can work following two different mechanisms: (1) enhancing radical activity or (2) quenching ROS inside cell compartments. In the first case, metal oxide NPs are able to convert superoxide ions into more reactive hydroxyl radical species via Fenton or Fenton-like reactions. The excess of superoxide ions is able to oxidize the viral proteins and the genetic material and therefore efficiently block the infection. This approach has been reported using ZnO NPs.117,118 In these studies, ZnO NPs have been successfully applied for the treatment of H1N1 influenza virus and Herpes simplex virus. The preliminary results showed that PEGylated ZnO particles were able to efficiently reduce the viral infection with IC50 similar to acyclovir. More importantly, toxicity of ZnO was modulated by functionalization with PEG, which allowed a higher colloidal stability and a more controlled release of Zn2+ ions to catalyze ROS formation. Indeed, ROS (e.g., superoxide and hydroxyl radicals) produced by the NPs should be highly reactive and should not only damage the exogenous biological molecules but also attack different cell compartments. Overproduction of ROS may reduce virus spread but also induce cell death. Interestingly, this approach is applicable only at the early infection stage (after 1 h incubation of the host cells with a virus), but it loses its activity at later time points. These experimental evidences suggest that materials capable of triggering the formation of ROS are essential in the first phase of the viral replication, most probably inducing the arrest of the viral DNA polymerases, which is active in the first 1–3 h of viral contamination.117,118 This specific mechanism of action restricts ZnO NPs to an application only at the early stage of infections. However, the same approach with other metals able to induce Fenton or Fenton-like reactions (e.g., Fe, Cu, and Mn) has not been reported yet. The choice of proper capping agents may allow to control the metal ion release and thus tune the ROS-mediated antiviral activity. We would like to take an advantage here to suggest the growth of the antiviral research in this direction.
Another successful approach relies on the reduction of ROS concentration in host cells. ROS scavenging is able to alleviate the toxicity of the infection enhancing cell viability, giving time to start its endogenous antiviral mechanisms. So, this approach may both block infection and ensure host cell survival. In this context, selenium NPs (SeNPs) have been extensively studied for their antiviral activity. The mechanism of action of these NPs relies on the quenching of the radicals into host cells due to the infection, stopping the mitochondria depolarization and the consequent apoptotic cascade.119 Additionally, SeNPs can also adsorb onto the viral capsid sensibly reducing their infectivity. SeNPs can be prepared via classical mixing of selenium salt precursors in the presence of a reducing agent. More recently, SeNPs have been instead biosynthesized from Actinobacteria showing good stability and capacity to inhibit Dengue virus in vitro.120 Moreover, SeNPs were used to carry different antiviral drugs including zanamivir,121 oseltamivir,122 amantadine,123 and ribavirin.119 Their functionalization with the desired drug can be easily achieved adding the molecule during their synthesis through the Se ion controlled reduction. These NMs have been applied for the treatment of H1N1 virus. Notably, SeNPs with ribavirin (administered via intranasal absorption every 24 h for 3 days) showed that infected mice had much less alveolar collapse and perivascular and peribronchiolar edema, compared to the group challenged with the virus (Figure 13).119
Figure 13  In vivo antiviral efficiency of SeNPs functionalized with ribavirin (Se@RBV). (a) Mice infected by H1N1 virus were treated with physiological saline (Mock), RBV, SeNPs, or Se@RBV. (b) H&E and tunnel staining showing that Se@RBV-treated mice displayed reduced lung damages compared to Mock. Reproduced with permission from ref (119). Copyright 2018 Dove Press Ltd. Due to their efficacy and low toxicity, SeNPs can be considered a useful material for the treatment of other viral diseases including SARS-Cov-2. As a matter of fact, oxidative stress as well as chronic inflammation may contribute to the aggravation of the Covid-19 symptoms and to the general spread of the infection.124 The use of SeNPs could eventually alleviate the toxicity of infected patients, giving time for the immune system to react against the contagion. However, the lack of preclinical studies on SeNPs, together the scarce knowledge of their biosafety and long-term toxicity, still remain the main challenges to tackle for clinical translation.